Bovine leukemia virus (BLV) is a blood-borne disease primarily transmitted mechanically by vectors and fomites. Straub, O. C. in Enzootic Bovine Leukosis and Bovine Leukemia virus, eds. Burny, A. & Mammerickx, M. (Martinus Nijhoff, Boston, Mass.), pp. 229-249 (1987). Approximately 30% of infected cattle develop persistent lymphocytosis, a polyclonal expansion of B lymphocytes. Ferrer, J. F. et al., J. Amer. Vet. Med. Assoc. 175:705-708 (1979); Kettmann, R. et al., Proc. Natl. Acad. Sci. USA 79:2465-2469 (1982) and Miller, L. D. et al., J. Nat. Cancer Inst. 48:423-428 (1972). The development of persistent lymphocytosis, in which the absolute number of lymphocytes (International Committee on Bovine Leukosis J. Nat. Cancer Inst. 41:243-263 (1968)) and the percentage of infected lymphocytes are dramatically increased (Burny, A. et al., Adv. Vet. Sci. Comp. Med. 32:149-170 (1988) and Kettmann, R. et al., Proc. Natl. Acad. Sci. USA 77:2577-2581 (1980)), markedly enhances the probability of transmission. Mammerickx, M. et al., Leuk. Res. 11:353-358 (1987).
The critical importance of persistent lymphocytosis to transmission of this blood-borne disease was demonstrated by experiments showing that less than 0.3 .mu.l of blood from cattle with persistent lymphocytosis was capable of transmitting BLV, while more than 1 ml of blood was necessary to transmit from infected cattle which did not have persistent lymphocytosis. Mammerickx, M. et al., Leuk. Res. 11:353-358 (1987). Moreover, vertical transmission from BLV-infected dams to their calves has been shown to be strongly correlated with persistent lymphocytosis. Agresti, A. et al., Amer. J. Vet. Res. 54:373-378 (1993).
In cattle, the ability to transmit BLV varies (Mammerickx, M. et al., Leuk. Res. 11:353-358(1987) and Weber, A. F. et al., Amer. J. Vet. Res. 44:1912-1915(1983)), and expression of antigen after in vitro culture has been shown to correlate with infectivity. Miller, L. D. et al., Amer. J. Vet. Res. 46:808-814 (1985). The level of BLV expression in the animal also may correlate with the probability of development of persistent lymphocytosis. Dropulic, B. et al., J. Virol. 66:1432-1441 (1992) and Cockerell, G. L. et al., Leuk. Res. 12:465-469 (1988). Moreover, persistent lymphocytosis is a strong risk factor for development of lymphoma. In 1-10% of the animals with persistent lymphocytosis, B cell clones undergo neoplastic transformation, leading to leukemia or lymphoma (Ferrer, J. F. et al., J. Amer. Vet. Med. Assoc. 175:705-708 (1979)), and cattle with persistent lymphocytosis are three times more likely to develop lymphoma than infected cattle without persistent lymphocytosis. Ferrer, J. F. et al., J. Amer. Vet. Med. Assoc. 175:705-708 (1979). BLV infection is thus a costly impediment to cattle production.
Currently, BLV is controlled by testing and slaughtering infected animals. Spread to newborn calves is reduced by raising them apart from their infected dams. However, in the United States, up to 66% of dairy herds are affected, and 13-48% of individual dairy cows carry the virus. Losses due to slaughter condemnation of cattle with tumors exceeds $42 million per year. In addition, the cattle industry suffers considerable losses due to inability to export cattle from BLV-positive herds to many foreign countries.
BLV, which causes the disease, is a retrovirus structurally similar to human T-cell leukemia viruses -I and -II (HTLV-I and -II). Burny, A. et al., Adv. Vet. Sci. Comp. Med. 32:149-170 (1988) and Miller, J. M. et al., J. Dairy Sci. 65:2194-2203 (1982). After initial infection, BLV expresses a doubly-spliced transcript which encodes the regulatory proteins, Tax and Rex. Sagata, N. et al., FEBS Lett. 192:37-42 (1985). Tax trans-activates the viral long terminal repeat (LTR) and cellular promoters, including those of c-fos and somatostatin, and may be involved in tumorigenesis. Katoh, I. et al., EMBO J. 8:497-503 (1989). Co-transfection experiments have demonstrated that Tax is necessary for viral expression in vitro. Willems, L. et al., EMBO J. 6:3385-3389 (1987); Rosen, C. A. et al., EMBO J. 5:2585-2589 (1986) and Derse, D., J. Virol. 62:1115-1119 (1988). Rex regulates the transition from early expression of the doubly-spliced transcript encoding regulatory proteins to the later expression of singly-spliced or unspliced transcripts which express the env gene or the gag and pol genes. Derse, D., J. Virol. 62:1115-1119 (1988). Recently, the 3' region of HTLV-I and BLV has been shown to encode RNA with alternative splice patterns which may express other regulatory proteins. Ciminale, V. et al., J. Virol. 66:1737-1 745 (1992); Koralnik, I. J. et al., Proc. Natl. Acad. Sci. USA 89:8813-8817 (1992) and Alexandersen, S. et al., J. Virol. 67:39-52 (1993).
Some success has been achieved in targeting RNA for cleavage and inactivation by means of antisense sequences. Haseloff, J. et al., Nature 334:585-591 (1988). Specific antisense sequences can be used to flank the hammerhead motif, first identified in plant RNA pathogens, which has been demonstrated to cleave the phosphodiester bond downstream of a GUC triplet. Also, ribozymes, particular forms of RNA, which can be targeted to attach to other RNAs and then enzymatically cleave them have been targeted to cleave a variety of sites of human immunodeficiency virus (HIV). Sarver, N. et al., Science 247:1222-1225 (1990); Heidenreich, O. et al., J. Biol. Chem. 267:1904-1909 (1992); Dropulic, B. et al., J. Virol. 66:1432-1441 (1992); Weerasinghe, M. et al., J. Virol. 65:5531-5534 (1991); Sioud, M. et al., Proc. Natl. Acad. Sci. USA 88:7303-7307 (1991) and Lo, K. M. S. et al., Virology 190:176-183 (1992).
It would thus be desirable to inhibit regulatory proteins in the BLV group of viruses. It would also be desirable to develop a ribozyme capable of cleaving rex/tax mRNA. It would further be desirable to develop expression plasmids containing such ribozyme DNA for transfection of BLV-infected cells and cleavage of BLV RNA. It would also be desirable to provide transgenic cattle which are resistant to BLV-induced persistent lymphocytosis and lymphoma.